Advances in Bioscience and Biotechnology
 Vol.3 No.4(2012), Article ID:21846,8 pages DOI:10.4236/abb.2012.34052

Efficient transcription of the larvicidal cry4Ba gene from Bacillus thuringiensis in transgenic chloroplasts of the green algal Chlamydomonas reinhardtii

Thanate Juntadech1, Kittisak Yokthongwattana2, Sithichoke Tangphatsornruang3, Yun-Kiam Yap1, Gerd Katzenmeier1, Chanan Angsuthanasombat1*

1Institute of Molecular Biosciences, Salaya Campus, Mahidol University, Bangkok, Thailand

2Department of Biochemistry and Center for Excellence in Protein Structure and Function, Faculty of Science, Mahidol University, Bangkok, Thailand

3National Center for Genetic Engineering and Biotechnology, Thailand Science Park, Pathum Thani, Thailand

Email: *chanan.ang@mahidol.ac.th

Received 18 March 2012; revised 19 April 2012; accepted 15 May 2012

Keywords: Chlamydomonas reinhardtii; Chloroplast Transformation; Inverted Repeats; Bt-cry4Ba Transcript; psbA Promoter

ABSTRACT

Unicellular micro-alga Chlamydomonas reinhardtii has been recognized as a promising host for expressing recombinant proteins albeit its limited utility due to low levels of heterologous protein expression. Here, transcription of the 3.4-kb mosquito-larvicidal cry4Ba gene from Bacillus thuringiensis in transgenic C. reinhardtii chloroplasts under control of the promoter and 5'-untranslated region of photosynthetic psbA gene was accomplished. Inverted repeats in chloroplast genomes of the host strain with deleted endogenous psbA genes were selected as recombination targets. Two transformant lines were obtained by dualphenotypic screening via exhibition of resistance to spectinomycin and restoration of photosynthetic activity. Stable and site-specific integration of intact cry4Ba and psbA genes into chloroplast genomes found in both transgenic lines implied homoplasmy of organelle populations. Achievement in co-transcription of cry4Ba and psbA transgenes revealed by RTPCR and Northern blot analyses demonstrates the sufficiency of this system’s transcription machinery, offering the further innovation for insecticidal protein production.

1. INTRODUCTION

Cry4Ba d-endotoxin, one of the four major insecticidal proteins produced from Bacillus thuringiensis subsp. israelensis (Bti), is specifically toxic against mosquito larvae of Aedes and Anopheles species that are vectors of widespread deadly contagious diseases, including dengue-viral hemorrhagic fevers, viral encephalitis and malaria (for reviews, see [1,2]). Achievement of over-expression of the recombinant Bti-cry4Ba toxin was at first reported in Escherichia coli [3]. A further effort was made until that time to manipulate this toxin into the cyanobacterial host, which can exist in an environment more akin to the target larvae [4]. Although the bacterial system holds some potential for producing this insecticidal protein, the expensive cost of industrial-scale production, particularly with regard to bacterial growth media, still hinders wide-scale usage of Bti toxins. Protein production in the plant expression system is inherently more cost-effective than that manufactured by bacterial fermentation [5]. However, time-consuming production and pollen-mediated gene flow are still the major drawbacks to this transgenic approach [6].

Single cell micro-alga Chlamydomonas reinhardtii, on the other hand, offers a compromise between the bacterial and plant systems in terms of production cost and timescale based on the fact that this microorganism makes use of natural resources, i.e., sunlight and CO2 for the photosynthetic growth as its generation time is approximately eight hours [7]. Moreover, the time spent from initial transformation to large-scale production is unexpectedly short, approximately six weeks for a flask scale and additional two more months for scaling up to several ten thousand liters [8].

Transgene expression in the chloroplast system thus far is the most attractive technology providing many advantages including the multiple copy number of chloroplast genomes with specific targeted integration via homologous recombination. In addition, the problem of gene silencing has not been observed in a transgenic chloroplast [9]. Moreover, in the perspective of biosafety, the chloroplast expression system provides an increase in transgene containment by the maternal inheritance as chloroplast genes are only inherited from the maternal parent while paternal chloroplast genes are selectively eliminated in young zygotes [10]. Together with the organism characteristics, these tremendous potentials make the chloroplast of micro-alga C. reinhardtii to be a promising bioreactor in terms of economic and commercial needs, although its utility is still limited owing to low levels of heterologous protein expression (for reviews, see [11]).

In order to construct the transformation vector harboring the cry4Ba gene to be efficiently expressed in the chloroplast of C. reinhardtii, controlling sequences of an algal strong promoter have to be first considered. It is interesting to note that translation of many chloroplast gene transcripts can be stimulated by light as the highest level of light induction has been reported for the photosynthetic psbA gene-encoded D1, a core protein of photosystem II [12]. Together with the fact that the combination of promoter/5'-UTR of psbA gene plays an important role in defining the levels of accumulation of recombinant proteins, whereas the identity of the 3'-UTR has little or no effect [13]. We therefore selected this powerful light-enhanced promoter in combination with its analogous 5'-UTR sequence to investigate the pliability of C. reinhardtii chloroplasts as biological factories capable of producing the prokaryotic cry4Ba gene product, even if there is still very limited knowledge on the translation of heterologous transcripts in these algal chloroplasts [11].

2. MATERIALS AND METHODS

2.1. Construction of C. reinhardtii Chloroplast Transformation Vectors

A transformation vector containing an antibiotic selection marker (aadA, encoding aminoglycoside adenyltransferase which confers resistance to spectinomycin treatment) and a photosynthetic gene (psbA) was constructed for screening transformants via both spectinomycin resistance and photosynthetic complementation. A right-arm recombination fragment (RF, NCBI_005353, bases 146,986 to 148,500), amplified from C. reinhardtii chloroplast genomes by RF-f and RF-r primers (Table 1), was introduced into pBluescriptII KS+ (Invitrogen) at SalI/ApaI sites, generating pCH1 (Figure 1, left panel).

Concurrently, an intermediary plasmid (p4Bds) containing the cry4Ba expression cassette was constructed (Figure 1, middle panel). A 3.4-kb EcoRI (fill-in with T4 DNA polymerase)/XbaI fragment of cry4Ba from pMU- 388 was ligated with a 3.5-kb NcoI (fill-in)/XbaI fragment of linearized p546 (the chloroplast expression vector, as

Table 1. Primers used in manipulation and verification of the cry4Ba transgene in c. reinhardtii chloroplasts.

was obtained from the Chlamydomonas Resource Center, www.chlamycollection.org) which contains the promoter, 5'-UTR and 3'-UTR of psbA. In order to facilitate ligation of the psbA-cry4Ba expression cassette into the chloroplast transformation vector, the flanking restriction sites were changed to SalI by subcloning into pUC18ds in which the original pUC18 EcoRI site was mutagenized to SalI (EcoRI∆SalI) via PCR-based mutagenesis (Stratagene) using the pUC18ds primer set (Table 1). In this process, a 4.2-kb KpnI (fill-in)/SacI fragment of the psbA-cry4Ba expression cassette was ligated with the 2.6-kb SmaI/SacI fragment of plasmid pUC18ds generating p4Bds. Subsequently, the cry4Ba expression cassette from p4Bds was excised and ligated to the SalI site of pCH1, yielding pCH2 (Figure 1, middle panel).

Finally, a 4.5-kb fragment, composed respectively of a left-arm recombination fragment (LF, NCBI_005353, bases 137,761 to 138,969) and two selectable expression cassettes consisting of the photosynthetic psbA-cDNA and the antibiotic-resistant gene, aadA, was amplified

Figure 1. Schematic diagram showing strategy for constructing C. reinhardtii chloroplast transformation vectors carrying the cry4Ba gene. Subcloning of each fragment into the pBluescriptII KS + backbone; the amplified RF fragment (left panel), the excised cry4Ba expression cassette fragment (middle panel) and the combined fragment containing LF and two selectable marker cassettes (right panel) were consecutively ligated into the plasmid backbone generating pCH1, pCH2 and pCH3, respectively. ampr denotes the ampicillin-resistance gene. For clarity, only those restriction sites relevant to the construction are shown.

from pBA157 template [14] by using LF-f and rbcL/3'- UTR-r primers (Table 1). The amplified product was subsequently introduced into pCH2 at NotI/SacII sites downstream of the cry4Ba expression cassette, generating the 13-kb chloroplast transformation vector, pCH3 (Figure 1, right panel).

2.2. C. reinhardtii Strains and Culture Conditions

C. reinhardtii wild-type (CC-400) and non-photosynthetic psbA-deleted (CC-744) strains were obtained from the Chlamydomonas Resource Center. Both strains were grown in TAP (Tris-acetate-phosphate) enriched medium, pH 7.0 [15], in a constant-illumination rotary shaker at 100 rpm, 25˚C with light intensity of ~50 µmol photons/ m2∙sec. Cells at late-log phase were concentrated by centrifugation until reaching an approximate concentration of 107 cells/ml. A 5-ml aliquot of the concentrated cells was spread onto a TAP agar plate containing spectinomycin (final concentration of 100 µg/ml) and then allowed to air-dry under sterile conditions.

2.3. Chloroplast Transformation in C. reinhardtii

Coating of gold particles (0.1 μm, Bio-Rad) with the chloroplast transformation vector (pCH3) was performed according to a modified version of a previously described procedure [16]. Chloroplast transformation was achieved via particle bombardment using a Heliumdriven apparatus (a PDS-1000/He, Bio-Rad), delivered at 900-psi pressure under 25-inches Hg vacuum and 9-cm target distance. After screening via spectinomycin resistance conferred by aadA expression cassette [17], C. reinhardtii transformant lines were subsequently verified for the recovery of photosynthesis by growing under high-salt (HS) minimal medium [18].

2.4. Genomic DNA Extraction and PCR Identification of Specific Integration

Total genomic DNA was extracted from C. reinhardtii cells using the CTAB (cetyltrimethylammonium bromide)- based protocol which was modified from the method of Murray and Thompson [19]. Briefly, after the cell lysate was treated with proteinase K (~0.20 mg/ml, Invitrogen) in Tris-EDTA buffer (pH 8.0) containing 6% SDS at room temperature (25˚C) for 15 min, the supernatant was separated by centrifugation and then incubated with DNA extraction buffer (10% CTAB, 0.7 M NaCl) at 65˚C for 10 min. The CTAB-extracted DNA was treated with phenol/chloroform/isoamylalcohol (25:24:1) extraction followed with two-time chloroform extraction and was recovered from aqueous phase by isopropanol precipitation.

PCR reactions (25 ml) containing total genomic DNA template (50 ng) and a pair of specific primers (5 pmol), psbA-f/L2, aadA-f/aadA-r and R1/4B-ORF (Table 1) in respectively generating products of recombinant 1.0-kb psbA, 0.4-kb aadA and 3.4-kb cry4Ba fragments, were initially performed. Specific localization of the delivered transplastomic genes was further verified by additional reactions employing two pairs of primers (i.e. L1/L2 and R1/R2, see Table 1) by which the generated PCR products would span left and right recombination targets, respectively. PCR reactions were performed for 30 reaction cycles (98˚C, 55˚C, 72˚C) using Phusion DNA polymerase (Thermo Scientific).

2.5. Semi-Quantitative RT-PCR Analysis

Total RNA was extracted from C. reinhardtii cells using the RNeasy plant mini kit (Qiagen) and then subjected to DNase treatment (RQ1 RNase-free DNase, Promega). 250 ng of DNA-free RNA and 10 µM of cry4Ba-specific R1 primer were used in reverse transcription reaction catalyzed by SuperScriptTM II reverse transcriptase (Invitrogen) in the presence of RNase inhibitor (RNaseOUTTM, Invitrogen). 1 µl of a 1:10 dilution of respective cDNA was used as template for the subsequent PCR reaction using primeSTAR Max DNA polymerase (Takara) with 4B-ORF and R1 primers (see Table 1) that bind specifically to start and stop codons of the cry4Ba gene, respectively.

2.6. Northern Hybridization Analysis

Large amounts of total RNA from chloroplasts were achieved by means of the ATA (aurintricarboxylic acid) large-scale RNA extraction method [20]. For Northern blotting, probes specific to cry4Ba (4B probe) and psbA (D1 probe) which are labeled with digoxigenin-11-dUTP (DIG, Roche) were PCR amplified by using primer sets for 4B (4B-946/4B-ORF) and D1 (psbA-f/psbA-r) probes, respectively (Table 1). After separation on denaturing formaldehyde/agarose gel, RNA was transferred onto a nylon membrane and then hybridized with DIG probe. Target fragments were localized by Anti-DIG (Roche) linked to alkaline phosphatase (AP) and light emission from the enzymatic dephosphorylation of chemiluminescent substrate (CDP-Star, Roche) was detected by X-ray film.

2.7. Protein Extraction and Western Blotting

Total cell and chloroplast protein extracts were prepared as previously described [21]. For Western blot analysis, proteins were fractionated on 10% SDS-PAGE, transferred to a nitrocellulose membrane and probed with the specific primary monoclonal antibody (2F-1H2 mouse antibody against the Cry4Ba-domain III fragment [22]. ECL (enhanced chemiluminescent) immunoblotting system (Pierce) was used to detect immunoreactive proteins with AP-conjugated goat anti-mouse IgG secondary antibodies.

3. RESULTS AND DISCUSSION

3.1. Establishment of Stable Chloroplast Transformant Lines

To promote cry4Ba gene expression in the C. reinhardtii chloroplast, the 3.4-kb cry4Ba coding sequence was incorporated between the psbA promoter/5'-UTR and the 3'-UTR. As with the Bti-cry4Ba gene, the psbA transgene is also transcriptionally regulated by the identical promoter and terminator sequences (Figure 1, right panel). In order to prevent the problem of deletion-looping out recombination [23], cry4Ba and psbA expression cassettes were assembled in the reverse orientation in the chloroplast transformation vector-pCH3 (Figure 1, right panel). It should be noted that both expression cassettes were placed between two integration regions which derived from sequences located within the inverted repeat regions of the C. reinhardtii chloroplast genome (Figure 1, right panel). Double copies of transgene integration into the cognate sequence in the chloroplast genome were thus expected.

Dissimilar to the gene expression of both cry4Ba and psbA transgenes, control of expression the bacterial aadA gene via the combination of atpA promoter/5'-UTR and rbcL 3'-UTR was also chosen instead since its analogous expression cassette has been successfully employed for this recombinant protein expression in C. reinhardtii chloroplasts [14].

Upon initial screening for the C. reinhardtii transgenic lines via resistance to spectinomycin treatment, only a few putative lines were recovered. The low efficiency of this chloroplast transformation may possibly be due to the fact in C. reinhardtii that recombination events within the inverted repeats of chloroplast genomes occur preferentially inside the 0.7-kb sequence spanning immediately the 3'-end of psbA gene known as hotspots [24]. Given that the mutant host strain used in our work was originally knocked out the whole set of psbA gene including the 0.7-kb hotspots, the remaining regions might therefore deteriorate the frequency of recombinetion events and hence decrease in the number of transgenic lines.

Further phenotypic recombinant screening revealed that two transformant lines, T1 and T2, showed the recovery of photosynthetic activity by means of scalability in HS minimal medium. It is noteworthy that the benefits of additional screening via photosynthetic psbA gene restoration would be very helpful in ruling out these transformant lines that may have resulted from spontaneous mutation induced by frequent drug screening [25]. It is also interesting to note that both spectinomycinresistance and restored-photosynthetic phenotypes for these two lines remained stable since both T1 and T2 lines have been monthly subcultured for more than a year on fresh TAP agar containing antibiotic spectinomycin. This indicates that the transformation vector was stably integrated into the chloroplast genome given that the co-existing plastid vector would become genetically incomepatible and be eventually eliminated [26].

3.2. Specific Integration of Intact Transgenes in Chloroplast Genome and Homoplasmy

Upon initial PCR identification via individual transgenespecific primers, single bands of each corresponding gene, 1.0-kb psbA, 0.4-kb aadA and 3.4-kb cry4Ba, were ably amplified from the total genomic DNA of both lines (T1 and T2) as shown in Figure 2(a). To further identify whether the transgenes are specifically integrated into the target inverted repeats of chloroplast genomes, two pairs of specific PCR primers, L1/L2 and R1/R2 which respectively bind to the psbA and cry4Ba transgenes, and to their corresponding adjacent endogenous chloroplast genes, were employed (Figure 2(b)). As can be seen that a 2.3-kb band was produced from the L1/L2 amplification by which the L1 primer annealed to the endogenous chloroplast chlN gene and the L2 primer annealed to the 3'-end of psbA transgene. In addition, a 5.1-kb band was amplified from the annealing of R1 and R2 primers to the 5'-end of the cry4Ba transgene and the endogenous chloroplast rrn5 (Figure 2(c)). These results indicate that both transgenes were entirely integrated into C. reinhardtii chloroplast genomes, further suggesting that the two identical psbA promoter sequences used in both cry4Ba and psbA expression cassettes did not cause a deletion-looping out phenomenon.

As mentioned earlier that the two transgenic lines stably exhibited both spectinomycin-resistance and recovered-photosynthetic phenotypes that are offered respectively by aadA and psbA transgenes. Therefore, this phenotypic stability along with the gene integrity of the chloroplast transgenes would most likely lead to homoplasmy of organelle populations as incompatible co-existing plastid molecules would be segregated to yield

Figure 2. Transgene integration into C. reinhardtii chloroplast genome. (a) PCR analysis of all three recombination fragments (1.0-kb psbA, 0.4-kb aadA and 3.4-kb cry4Ba, as indicated above each corresponding figures) amplified from total genomic DNA extracted from both transformant lines, T1 and T2. NC denotes a non-transformant line CC-744 used as a negative control. Only those sizes of standard markers, M1 (100-bp DNA ladder) and M2 (BstEII-digested λ DNA) relevant to amplified products are shown; (b) Schematic representation of the expected left inverted repeat (IRb) region in the transgenic chloroplast genome as a result from specific integration of the pCH3 transformation vector. Binding sites of individual primer pairs, L1/L2 and R1/R2, and their correlated spanning products are shown under the map. Corresponding locations of D1 and 4B probes used in Northern blotting are denoted by black bars; (c) PCR identification of specific integration of both psbA and cry4Ba transgenes, showing 2.3-kb and 5.1-kb products amplified from both transformant lines, using L1/L2 and R1/R2 primer sets, respectively.

homoplasmic isolates after a long period repeatedly subculture under selective conditions [27].

3.3. High Yield and Integrity of cry4Ba Transcripts in Transgenic Chloroplasts

To verify that the 3.4-kb cry4Ba transgene which was wholly integrated in the chloroplast genome and placed under the photosynthetic psbA promoter was ably transcribed, RT-PCR and Northern blotting were performed. For RT-PCR analysis, the results revealed that the cry4Ba gene in both T1 and T2 lines is efficiently and completely transcribed as indicated by the presence of a 3.4-kb high-yield product comparable to that directly amplified from the control vector template (Figure 3). It is noteworthy that this 3.4-kb corresponding band was absent in the RT-negative control, confirming that the RT-PCR product was derived from the DNA-free cry4BamRNA template. In addition, this single full-length RTPCR product of 3.4 kb could be an indicative of the transcript integrity.

Consistent with the RT-PCR results, Northern blotting (NB) also revealed a major ECL-positive band of ~3.4 kb in the total RNA extracts of both T1 and T2 lines when hybridized with the specific cry4Ba probe (Figure 4, right panel), further strengthening the efficient transcription of the recombinant cry4Ba gene in transgenic chloroplasts. It is worth mentioning that an intense NB band of ~1.0-kb was specifically revealed in both transgenic lines when probed with the psbA-D1 fragment (Figure 4, left panel). The data thus established that algal chloroplasts have adequacy of basal transcription factors utilized to efficiently co-transcribe the cry4Ba and psbA transgenes driven by each individual psbA-controlling cassette.

3.4. Deficient Production of the Recombinant cry4Ba Protein in Transgenic Chloroplasts

Attempts were also made to examine the expression of

Figure 3. RT-PCR analysis of cry4Ba transgenic transcripts in both C. reinhardtii transgenic lines, T1 and T2. PC and NC denote positive and negative PCR control reactions, respectively, as PC contains the pCH3 template while NC omits a corresponding template. Neg-RT represents a control RT-PCR reaction with reverse transcriptase enzyme omitted. Relevant sizes of DNA marker (M, Gene ladder fast 2) are shown.

Figure 4. ECL-autoradiograms of Northern blots containing total RNA extracted from C. reinhardtii transgenic lines, T1 and T2, showing the accumulation of the two transgenic transcripts, i.e. ~3.4-kb cry4Ba (right panel, as detected by 4B probe) and ~1.0-kb psbA (left panel, as detected by D1 probe). NC denotes a negative control as RNA was extracted from strain CC-744. Total RNA from the CC-400 wild-type strain was used as a positive control for the synthesis of an endogenous psbA transcript.

the 130-kDa Cry4Ba recombinant protein in transgenic chloroplasts of T1 and T2 lines which show efficient transcription of this intact transcript as demonstrated earlier (see Figure 3). As assessed by ECL immunoblotting probed with the specific Cry4Ba-domain III monoclonal antibody, the results revealed no demonstrable accumulation of this recombinant protein under the conditions used (data not shown). These results therefore make a note of a poor correlation between protein accumulation and high-yield transcript production in the C. reinhardtii chloroplast as also noticed previously [28,29]. However, the reasons why this particular heterologous transcript is defectively translated in the transgenic chloroplast are still unclear at present.

A number of studies have revealed that translational efficiency is a rate-limiting step for transgene expression in C. reinhardtii chloroplasts, and some have demonstrated that the bias seen in codon usage has a profound effect on recombinant protein accumulation in the algal chloroplasts (for reviews, see [11]). Here, we thus evaluated the codon usage of the Bti-Cry4Ba protein-coding gene to check its preference for the utilization of synonymous persistent codons in comparison with that of the C. reinhardtii chloroplast genome (www.kazusa.org. jp/codon). Although the patterns of synonymous codon usage in both the bacterial cry4Ba transgene and the C. reinhardtii chloroplast genome are quite similar as almost all codons ending in A or U are preferred, there is a strong bias in the Cry4Ba-coding gene towards the use of glycine GGG and histidine CAC codons (see Table 2). It is therefore possible that the deficient translation of the high-yield cry4Ba transcript in transgenic chloroplasts could perhaps be due to biases seen in glycine and histidine codons used in this recombinant protein-coding gene. Hence, further studies via codon optimization of

Table 2. Comparison of the bias usage between the Bti-cry4Ba transgene and the C. reinhardtii chloroplast genome (Kazusa database, www.kazusa.org.jp/codon) noticed in glycine and histidine codons.

this non-native gene are of great interest since a codonoptimized cry4Ba gene might be indeed a requirement for improving the heterologous production of the Cry4Ba insecticidal protein in C. reinhardtii chloroplasts, although some other unmodified Bt-cry genes, i.e. cry1Ab, cry1Ac and cry2Aa2, have been effectively expressed with high levels of insecticidal protein accumulation in plant chloroplasts (for reviews, see [22]). Nevertheless, another possibility that the Cry4Ba-coding sequence per se, besides codon bias, might be crucial for translation efficiency in transgenic chloroplasts remains to be investigated.

4. ACKNOWLEDGEMENTS

We gratefully acknowledge Dr. Jun Minagawa for providing plasmid pBA157. Special thanks to Drs. Hidetaka Hori, Keiichi Okazaki and Kimiko Itoh (Niigata University, Japan) for technical advice. This work was funded in part by grants from BIOTEC and Shell foundation. Royal Golden JubileePhD scholarship from Thailand Research Fund (to T.J.) is acknowledged.

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NOTES

*Corresponding author.

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